NSP酶共展示体系的构建及在肉鸡小麦日粮中的协同降解机制

Wheat has not been large-scale application in feed ingredients because it contains significant quantities of non-starch polysaccharides (NSP) which have various anti-nutritional effects in monogastric animals. NSP enzymes are commonly added to wheat-based diets to counter the possible anti-nutritional effects by degrading NSP. Previous reports showed that multi-enzyme preparations including xylanase, β-glucanase and pectinase exerted synergistic effects in promoting the degradation of wheat. However, NSP enzymes from various sources showed such a difference in performance and effects and the mechanism of their synergistic effects remains unclear. In this study, NSP multi-enzyme preparations will be produced by yeast cell surface display technology and trifunctional minicellulosomes. Subsequently, these enzymes will be added to samples of wheat that have been discarded starch. Herein, the aim of this study is to investigate the mechanism of their synergistic effects. The occurrence time of synergy, the type of synergy and the degree of synergy will be determined first. Then, the changes of substrate surface ultrastructure will be detected by local ultrasturctural analysis during the process of synergetic degradation of wheat. Meanwhile, different levels of wheat overall measurement techniques will be applied to analyze the changes of substrate overall structure when wheat are dealt by NSP multi-enzyme preparations. Furthermore, effects of NSP multi-enzyme preparations on growth performance, digesta viscosity and gut microbiota of broiler chickens will be investigated. According to the results to be researched on above-mentioned, we can survey the possible synergistic mechanism of NSP multi-enzyme preparations and explore key factors for efficient degradation of NSP. This could offer the theory basis for wheat application in feed ingredients.

小麦由于含有大量NSP而具有一定的抗营养特性，限制了其在饲料中的广泛应用。向小麦日粮中添加NSP酶可降解小麦中的NSP从而降低抗营养作用。已有研究证实木聚糖酶、β-葡聚糖酶和果胶酶的复合酶在降解小麦NSP时存在协同作用，但不同来源的酶性能差异大，作用效果层次不齐，且协同作用机理尚不清楚。本研究在前期获得三种耐热NSP酶的基础上，利用酵母表面展示技术和纤维小体体系制备性能优良的共展示酶，将其作用于去淀粉小麦样品，分析协同作用发生的方式、时间、强度等，同时采用超分子结构表面分析技术观察共展示酶和单酶降解过程中底物表面超微结构的区别与变化，测定小麦样品在共展示酶和单酶降解过程中整体结构发生变化的程度，结合共展示酶和单酶对肉鸡生产性能、肠道食靡黏度和肠道菌群影响的差异，系统探讨三种酶对小麦NSP的协同降解机制，寻找小麦NSP高效降解的关键因素，为小麦在饲料中的广泛应用提供理论基础。

小麦是一种重要的家禽饲料原料，然而由于小麦中含有大量的非淀粉多糖（NSP）使其具有一定的抗营养特性，向小麦日粮中添加NSP酶复合酶能有效降解小麦中的NSP，但降解效率不仅受各单酶性能和复配的影响，也与各酶之间的协同作用密切相关。本项目根据小麦中NSP的组成，针对性的筛选出三种耐热NSP酶，利用酵母表面展示技术将三种酶在毕赤酵母中进行了展示表达，并对所使用的锚定蛋白进行了优化，成功获得了三种NSP酶的展示酶。在此基础上进一步构建获得了重组菌X33/DEP，该菌可同时展示表达三种NSP酶，实现了“一菌产多酶”，揺瓶水平该重组菌产三种酶的最大酶活分别为木聚糖酶酶活12583 U/g、葡聚糖酶酶活1984 U/g、果胶酶酶活5932 U/g。将三种NSP酶展示酶用于小麦麸NSP的降解，结果显示无论是同步性协同方式还是连续性协同方式，葡聚糖酶EGⅡ和木聚糖酶DSB的协同作用效率强于果胶酶PG5和木聚糖酶DSB，而三种酶共同作用时协同效率最高。此外与同步性协同方式相比，连续性协同方式并没有使两种酶或三种酶呈现出更高的协同效率。在连续性协同方式分析中，木聚糖酶DSB先作用的方式的协同因子（DS）均大于木聚糖酶DSB后作用的方式，且水解后产物分析的结果显示，与单独木聚糖酶DSB相比，三种酶同时作用时葡萄糖生成量的增加高于木寡糖生成量的增加，可能原因为木聚糖酶降解木聚糖后增加了麦麸中木质纤维的孔隙度，使得另外两种酶更易接近底物从而更有效的发挥水解作用。在肉鸡生长过程中，与单独添加木聚糖酶DSB展示酶相比，添加三种NSP酶共展示酶进一步提高了肉鸡的生长性能，增加了饲料利用率。本项目的研究结果为简化复合酶的生产、制备性能优良的NSP复合酶及小麦麸中NSP的有效降解提供了一定的理论依据。